Arsenic, a naturally occurring element, is a world-wide public health concern affecting millions of people. In humans, arsenic is primarily metabolized by AS3MT to yield both trivalent and pentavalent mono- and less toxic dimethylated arsenicals. Variability in arsenic biomethylation and the resulting changes of arsenic metabolite profiles contribute to the differential susceptibility of individuals to the toxc and carcinogenic effects of arsenic. Evidence suggests that AS3MT may not be the only methyltransferase in human cells and tissues that is capable of methylating arsenic and modifying its toxicity. Given the central role of arsenic metabolism in arsenic-induced toxicity and carcinogenicity, there is a critical need to identify other potential arsenic methyltransferase, which may contribute to arsenic bioactivation and detoxification. Our recent report demonstrated for the first time that N-6 Adenine-Specific DNA Methyltransferase 1 (N6AMT1), a new methyltransferase, can biotransform highly toxic monomethylarsonous acid (MMAsIII) to less toxic dimethlated arsenicals (DMAs), conferring resistance of human cells to iAsIII and MMAsIII. Based on these observations, our overall hypothesis is that N6AMT1 participates in arsenic metabolism and varied expression and altered function of N6AMT1 can modify arsenic metabolic profile, and subsequently alter genetic susceptibility of individuals and the tissue-specific sensitivities to arsenic-induced health hazards. However, before this hypothesis can be addressed in human population-based studies, there is a need to address fundamental gaps in our understanding the role of N6AMT1 in arsenic metabolism, including: 1) Substrate specificity, that is which form of arsenicals (oxidized, methylated or thiol-containing arsenicals) can be metabolized by N6AMT1? 2) What are the kinetic parameters for human N6AMT1- and AS3MT-mediated arsenic metabolism? 3) Can N6AMT1 compensate for low AS3MT activity in protecting against arsenic toxicity? 4) What is the relative abundance of N6AMT1 protein in human target and non-target tissues as well as cancer types? The primary objectives of this application will address these questions by characterizing and comparing the kinetic parameters for arsenic biomethylation by N6AMT1 and AS3MT and investigating their interactive effects on arsenic metabolism and the resulting toxicological responses to arsenic (Aim 1&2). A secondary objective is to determine the relative level of N6AMT1 protein in human target and non-target tissues and across cancer types (Aim 3). The contribution of this study is significant because once it is understand the role and the relative contribution of N6AMT1 to arsenic biomethylation; it will significant advance our understanding of N6AMT1-mediated arsenic metabolism and its potential role in differential susceptibility of individuals and the tissue-specific sensitivities o arsenic toxicity, and help to establish the rationale for future mechanistic and population based studies.